Bombyxamycins A and B, Cytotoxic Macrocyclic Lactams from an Intestinal Bacterium of the Silkworm Bombyx mori

Article abstract of DOI:10.1021/acs.orglett.9b00384

Bombyxamycins A and B (1 and 2) were discovered from a silkworm gut Streptomyces bacterium. Spectroscopic analysis and multiple-step chemical derivatization identified them as 26-membered cyclic lactams with polyene features. Bombyxamycin A showed significant antibacterial and antiproliferative effects. The bombyxamycin biosynthetic gene cluster was identified by genetic analysis. Gene deletion experiments confirmed that the cytochrome P450 BomK is responsible for the generation of 2, which unprecedentedly bears tetrahydrofuran in its macrocyclic ring.

Full text of DOI:10.1021/acs.orglett.9b00384

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Bombyxamycins A and B, Cytotoxic Macrocyclic Lactams from an
Intestinal Bacterium of the Silkworm Bombyx mori
Yern-Hyerk Shin,† Ji Yoon Beom,‡ Beomkoo Chung,§ Yoonho Shin,† Woong Sub Byun,† Kyuho Moon,†
Munhyung Bae,† Sang Kook Lee,† Ki-Bong Oh,§ Jongheon Shin,† Yeo Joon Yoon,*,‡
and Dong-Chan Oh*,†
†Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea
‡Department of Chemistry and Nanoscience, Ewha Womans University, Seoul 03760, Republic of Korea
§Department of Agricultural Biotechnology, College of Agriculture & Life Sciences, Seoul National University, Seoul 08826,
Republic of Korea
S
* Supporting Information
ABSTRACT: Bombyxamycins A and B (1 and 2) were discovered from a silkworm gut Streptomyces
bacterium. Spectroscopic analysis and multiple-step chemical derivatization identified them as 26-
membered cyclic lactams with polyene features. Bombyxamycin A showed significant antibacterial and
antiproliferative effects. The bombyxamycin biosynthetic gene cluster was identified by genetic analysis.
Gene deletion experiments confirmed that the cytochrome P450 BomK is responsible for the generation of
2, which unprecedentedly bears tetrahydrofuran in its macrocyclic ring.
or the past 10 years, insect symbionts have been
from Lepidopteran insect-associated microbes. Here, we report
Frecognized as promising chemical sources of bioactive the structure, biological activity, and genome-based biosyn-
small molecules.1 Chemical investigations of insect-associated
thesis of these new compounds.
Bombyxamycin A (1) was isolated as a yellow powder. A
molecular formula of C27H33NO3 was identified by high-
resolution fast atom bombardment mass spectrometry (HR-
FAB-MS). Based on the molecular formula, the unsaturation
microorganisms were initiated with insects belonging to the
orders Coleoptera and Hymenoptera. As representative
pioneering works, a novel antifungal polyene peroxide and
new polycyclic macrolactams were discovered from symbiotic
Streptomyces strains of the southern pine beetle, Dendroctonus
frontalis (Coleoptera).2 A new antifungal peptide, dentiger-
umycin A, was isolated from a Pseudonocardia strain in the
fungus-growing ant Apterostigma dentigerum (Hymenoptera).3
Subsequent chemical studies mainly focused on microbes
associated with Coleopteran and Hymenopteran insects,
leading to structurally novel organic compounds with
pharmaceutical potential.4−10 However, even though Lepidop-
tera is the second largest order after Coleoptera,11 the
chemistry of microbes associated with Lepidopteran insects
has rarely been studied. Only a proteinaceous toxin, identified
as an antibiotic modulating the gut microbiota from a
symbiotic bacterium, Enterococcus mundtii, in the cotton
leafworm Spodoptera littoralis was discovered in 2017.12 The
silkworm Bombyx mori, including its microbiota, is the most
heavily studied Lepidopteran insect because of its industrial
and medicinal importance.13 The paucity of chemical
investigations of microbes in B. mori led us to focus on
silkworm gut bacteria. In our chemical examination of gut
microorganisms of B. mori larvae, a bacterial strain,
Streptomyces sp. SD53, was thought to produce a new series
of polyene compounds based on ultraviolet (UV) spectro-
scopic data (λmax 323 and 383 nm). Large-scale culture of the
strain and subsequent chromatographic purification allowed for
the discovery of previously unreported macrocyclic lactams,
bombyxamycins A and B (1 and 2), as the first small molecules
1
number of 1 was deduced as 12. Combined analysis of H
(Table S1) and HSQC NMR data of 1 showed the existence of
one amide proton (δH 7.97) and 17 olefinic methine protons in
the downfield region (δH 7.45−5.23) (Table S1). One oxygen-
bound methine proton (δH 4.04), one hydroxyl proton (δH
4.77), six protons (δH 3.46, 2.86, 2.46, 2.31 (2H), and 2.30)
belonging to three methylene groups, one aliphatic methine
proton (δH 3.12), and two methyl group protons (δH 1.64 and
1
0.94) were also identified by H and HSQC NMR data. The
13C NMR spectral data revealed that 1 bears two carbonyl
carbons (at 199.8 and 166.3 ppm), 18 olefinic carbons (δC
140−120), one oxygen-bound carbon (δC 68.5), and six
aliphatic carbons, including two methyl groups (δC 17.9 and
12.5). On the basis of the NMR and UV spectroscopic data, 1
was deduced to possess a couple of conjugated chromophores
with double bonds in its structure.
By interpretation of COSY NMR correlations of 1, three
discrete spin systems were identified. One of the spin systems
from C-2 could be successfully traced to C-7 by a series of
COSY correlations from H-2 (δH 5.55) to H-7 (δH 6.41).
1
Consecutive H−1H couplings from H-9 (δH 5.52) to H2-12
(δH 2.86 and 2.46) through H-10 (δH 2.31) and H-11 (δH
4.04) established the C-9−C-10−C-11−C-12 connectivity.
Received: January 30, 2019
© XXXX American Chemical Society
A
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Letter
The last spin system was assigned based on an array of COSY
signals from H-14 (δH 6.15) to 25-NH (δH 7.97). H3-2′ (δH
0.94) was connected to C-24 by H3-2′/H-24 (δH 3.12) COSY
correlations.
Figure 2. ΔδS−R values of (S)- and (R)-MTPA esters of (a)
bombyxamycin A (1) and (b) bombyxamycin B (2).
Scheme 1. Chemical Reactions for the Identification of the
a
Absolute Configuration at C-24 of 1
An HMBC correlation from H-2 to C-1 (δC 166.3)
established the connectivity between the olefinic carbon C-2
(δC 121.3) and the carbonyl carbon C-1. Analysis of HMBC
correlations from the methyl protons H3-1′ (δH 1.64) to C-7
(δC 140.1), C-8 (δC 135.1), and C-9 (δC 131.4) connected the
first and the second spin systems through the fully substituted
olefinic carbon C-8. Furthermore, the two-bond HMBC signals
from H2-12 and H-14 to C-13 (δC 199.8) indicated that the
carbonyl carbon C-13 was between the second and the third
spin systems, resulting in a pentaenone-bearing structure.
Because two carbonyl groups and nine double bonds explained
11 unsaturation equivalents out of 12, 1 was deduced to
possess a ring. Finally, a 25-NH/C-1 HMBC correlation
established the 26-membered monocyclic structure of 1
(Figure 1a). The double-bond geometries in 1 were clarified
a
ΔδS−R values in ppm are noted for 3a and 3b.
with Sanger’s reagent. The product, 3-(2,4-dinitrophenylami-
no)-2-methylpropanoic acid (3), was additionally derivatized
with (S)- and (R)-phenylglycine methyl ester (PGME) to yield
(S)- and (R)-PGME amides (3a and 3b) of 3.15 Discrete
analysis of 1H NMR spectral data of these PGME products (3a
and 3b) enabled the determination of the absolute
configuration of 3 as 2S based on ΔδS−R values, thus
establishing the 24R configuration of 1 (Scheme 1).
Bombyxamycin B (2) was obtained as a pale-yellow powder
by consecutive chromatographic isolation. The molecular
formula of 2 could be assigned as C27H31NO5 with an
unsaturation number of 13 by using HR-FAB-MS data.
Analysis of 1D and 2D NMR data of 2 (Table S1) revealed
that it possesses two carbonyl carbons, nine double bonds,
three oxygen-bearing sp3 carbons, and four aliphatic carbons,
including two methyl groups. Because 2 has one more
unsaturation equivalent than and the same numbers of
carbonyl carbons and double bonds as 1, it was deduced
that 2 must bear an additional ring. Examination of the COSY
NMR spectrum elucidated the two polyene spin systems (C-1
to C-7 and C-14 to 25-NH) that were consistently found in 1
(Figure 1b). The other spin system was composed of two
oxygen-bearing methine groups, and two hydroxyl groups were
identified by H-9/H-10, H-9/9-OH, and H-10/10-OH COSY
correlations. The HMBC correlation signal from H3-1′ to C-7,
C-8, and C-9 demonstrated C-7−C-8−C-9 connectivity. In
addition, 1H−13C correlations from H-10 to C-11 and from H-
12 to C-11, C-13, and C-14 allowed for the construction of a
long polyketide chain from C-1 to 25-NH. As shown in 1, a 26-
membered ring structure of 2 could be secured by HMBC
correlation between 25-NH and C-1 (Figure 1b).
Figure 1. Key COSY and HMBC correlations for constructing the
planar structure of (a) bombyxamycin A (1) and (b) bombyxamycin
B (2).
as 2Z,4Z,6E,14E,16Z,18Z,20E,22Z by measuring 3JHH values of
the olefinic protons (Table S1 and Figure S2). The 8Z
geometry was established by H-9/H3-1′ NOESY correlation.
The absolute configuration of C-11 of 1 was established by
the modified Mosher’s method.14 The secondary alcohol at C-
11 was derivatized with (R)- and (S)-α-methoxy-
(trifluoromethyl)phenylacetyl chloride (MTPA-Cl) to yield
(S)- and (R)-MTPA esters (1a and 1b) (Figure 2a). ΔδS−R
1
values were calculated by analysis of H and COSY NMR
spectra of 1a and 1b. The signs of ΔδS−R values were
consistently distributed, assigning the 11R configuration
(Figure 2a).
To determine the absolute configuration of C-24 in 1,
ozonolysis and acid hydrolysis of 1 were conducted (Scheme
1).7 After the sequential chemical reactions, the desired β-
amino acid (3-amino-2-methylpropanoic acid) was derivatized
The planar structure of 2 was then proposed by connecting
the last oxygen-bearing sp3 carbon, C-8 (δC 89.1), and the fully
B
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Letter
substituted, highly deshielded olefinic carbon C-11 (δC 167.6)
through an ether linkage to make one more ring based on the
molecular formula even though HMBC correlations indicating
the five-membered ether ring could not be observed because
C-8 and C-11 do not have a proton (Figure 1b). However, the
ether bond was further supported by 13C NMR experiments
for 2 in CD3OD and a 1:1 mixture of CD3OD/CD3OH
(Figure 3). Carbon signals that are located at the β-position of
niae ATCC 10031, Salmonella enterica ATCC 14028, and
Escherichia coli ATCC 25922). Compound 1 showed
significant inhibitory activity against P. hauseri (MIC = 0.5
μg/mL), S. enterica (MIC = 1 μg/mL), and S. aureus (MIC = 8
μg/mL), whereas 2 moderately inhibited only S. aureus (MIC
= 64 μg/mL) (Table S2). The antiproliferation activities of 1
and 2 were also evaluated in a panel of human cancer cell lines
representing lung cancer (A549), colon cancer (HCT116),
stomach cancer (SNU638), liver cancer (SK-HEP-1), breast
cancer (MDA-MB-231), and leukemia (K562) (Table S3).
Compound 1 displayed remarkable antiproliferation activities
against most of the tested cancer cell lines (IC50 = 0.39−1.76
μg/mL). However, 2 exhibited weak activity against only K562
cells, with an IC50 value of 37 μg/mL. Bombyxamycins A and
B exhibited no significant inhibitory effect (MIC ≥ 128 μg/
mL) against the tested pathogenic fungi (Candida albicans
ATCC 10231, Aspergillus fumigatus HIC 6094, Trichophyton
rubrum NBRC 9185, and Trichophyton mentagrophytes IFM
40996).
Approximately 8.1 Mb of draft genome sequence was
obtained from the bombyxamycin producer strain Streptomyces
sp. SD53. Analysis of the sequences with antiSMASH 4.017
(Table S4) revealed that the bombyxamycin biosynthetic gene
clusters contained six genes encoding polyketide synthases
(PKSs), eight characteristic enzymes involved in β-amino acid
synthesis, and several putative post-PKS tailoring enzymes
(Figure 4a and Table S5). The core macrolactam ring of
bombyxamycin is biosynthesized by a type I modular PKS,
BomP1−BomP6, which comprises a loading and 11 elongation
modules. These modules are consistent with the 26-membered
structure of 1 except for the presence of the ketoreductase
(KR) domain in module 6. However, a detailed sequence
analysis of the modules revealed that the ketoreductase domain
in module 6 is nonfunctional due to the absence of the highly
conserved KR active site motif and an NADP(H) binding
motif, which explains the ketone group at C-13 (Figure S32).18
In addition, the KR domain of module 7 is an A-type KR,
which contains a conserved tryptophan residue (“W” motif)
instead of the LDD motif in B-type KRs (Figure S32).19 This
sequence analysis is consistent with the chemically determined
11R configuration of 1. A β-amino acid starter unit (3-amino-
2-methylpropanoic acid) is predicted to be synthesized from
glutamic acid by glutamate mutase BomG/BomH, decarbox-
ylase BomI, acyl carrier protein (ACP) BomD, two ATP-
dependent ligases (BomE and BomJ), and acyltransferase
BomM in a similar way as the starter unit biosynthesis of
vicenistatin.20 After the polyketide chain is fully processed, we
propose that the terminal acyl group (alanine) is removed by
the L-amino acid amidase BomC before macrolactam
formation by the thioesterase domain of BomP1 (Figure 4b).21
Compound 1 appeared to be modified by post-PKS
modification enzymes to produce 2. Although the detailed
post-PKS modification route is unresolved, we propose a
pathway for 2 biosynthesis based on sequence analysis and the
deletion of one of the genes involved (Figure 4c). First, a
cytochrome P450 hydroxylase (BomK, BomN, or BomO)
catalyzes the hydroxylation at C-10 of 1. With the action of a
monooxygenase (BomA and BomB), the intermediate is
hydrated, and nonbonding electrons of the C-11 hydroxyl
group could attack C-8, forming a THF ring structure. To
identify the gene responsible for the C-10 hydroxylation step,
each gene encoding bomK, bomN, and bomO was inactivated
by in-frame deletion (Figures S33−S35). Compound 2
Figure 3. 13C NMR spectra of 2 in (a) CD3OD and (b) CD3OD/
CD3OH = 1:1.
exchangeable protons are doubled or broadened in a 1:1
mixture of CD3OD and CD3OH by a β-isotope effect.16 The
carbon peaks of C-9 and C-10 of 2 were broadened, whereas
C-8 and C-11 peaks were not affected by the mixed solvent,
indicating that C-8 and C-11 are oxygen bearing but not bound
to −OH (Figure 3). This result strongly supported the
elucidated structure of 2.
The relative configuration of the tetrahydrofuran (THF)
moiety was assigned by analysis of NOESY NMR data. H-7/H-
9 and H3-1′/H-10 NOESY correlations indicated that H-7 and
H-9 are in the same phase. In contrast, H3-1′ displayed a
NOESY correlation with H-10, indicating that these protons
are on the other side of the molecule. Therefore, the relative
configurations of the THF in 2 were established as
8R*,9S*,10S*. The geometries of the double bonds were
1
assigned mainly by H−1H coupling constants (Table S1 and
Figure S9). The H-12/H-10 NOESY correlation allowed for
the 12Z configuration (Figure S19).
The absolute configurations at C-8, C-9, and C-10 of 2 were
determined by the application of the modified Mosher’s
method. With a short derivatization time (5 min) and using
(R)- and (S)-MTPA-Cl, mono-(S)- and (R)-MTPA esters (2a
and 2b) of 2 were predominantly obtained rather than bis-
MTPA esters. Analysis of 1H and COSY NMR spectral data of
2a and 2b revealed that 10-OH in 2 was esterified. Calculation
of ΔδS−R values enabled the determination of the 10S
configuration. Based on the relative configurations, the
absolute configurations of C-8 and C-9 were assigned as 8R
and 9S (Figure 2b). The absolute configuration of C-24 was
determined as R by the same procedure mentioned above for
1.
Compounds 1 and 2 were biologically evaluated for
antibacterial activity (Table S2) against several human
pathogenic bacterial strains (Staphylococcus aureus ATCC
25923, Enterococcus faecalis ATCC 19433, Enterococcus faecium
ATCC 19434, Proteus hauseri NBRC 3851, Klebsiella pneumo-
C
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Letter
Figure 4. (a) Biosynthetic gene cluster for 1 and 2. (b) Schematic representation of predicted biosynthetic procedure for 1. Domains within each
module are represented by circles. The black circle indicates a domain that is predicted from the final structure and deletions in the active sites to
not be active. KS, ketoacyl synthase; AT, acyl transferase; DH, dehydratase; KR, keto reductase; ACP, acyl carrier protein: TE, thioesterase. (c)
Proposed biosynthetic pathway for producing 2 from 1.
production was not changed in the knockout mutants ΔbomN
and ΔbomO (Figures S36 and S37). However, the ΔbomK
mutant led to the selective loss of 2 production and the
contrasting accumulation of 1, confirming that this gene is
involved in 2 biosynthesis (Figure S38).
isms. This result indicates that expanding the chemical
investigation of microbes inhabiting insects to Lepidoptera
could lead to additional successful discoveries of unexplored
novel compounds with pharmaceutical potential.
ASSOCIATED CONTENT
* Supporting Information
Bombyxamycin A (1) is a structurally unique 26-membered
macrolactam bearing tetraenone and pentaenone. Sceliphro-
lactam from a mud dauber-associated Streptomyces is the most
similar compound with a 26-membered lactam ring.9 However,
sceliphrolactam exhibits different C-methylation and hydrox-
ylation patterns. Mirilactam B22 and FW05328-123 are
previously reported 26-membered macrocyclic lactams, but
their carbon skeletons differ by possessing a methyl group at
the α-position of the amide nitrogen, which indicates that their
biosynthesis starts with an α-amino acid unit rather than a β-
amino acid unit as in 1. Cyclamenol shares its β-amino acid
starting block with the bombyxamycins,24 but it possesses a 20-
membered cyclic lactam skeleton differently from the 26-
membered framework of 1 and 2. The structural novelty of 2 is
even higher because of the incorporation of a secondary ring,
THF, in the middle of the macrocycle. To the best of our
knowledge, this is the first bicyclic macrolactam with
substituted THF. The bombyxamycins are the first organic
molecules from Lepidopteran insect-associated microorgan-
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.9b00384.
Detailed experimental procedures and NMR data for
compounds 1 and 2 (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
*E-mail: joonyoon@ewha.ac.kr
*E-mail: dongchanoh@snu.ac.kr.
ORCID
Sang Kook Lee: 0000-0002-4306-7024
Jongheon Shin: 0000-0002-7536-8462
Yeo Joon Yoon: 0000-0002-3637-3103
Dong-Chan Oh: 0000-0001-6405-5535
D
DOI: 10.1021/acs.orglett.9b00384
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Notes
Letter
(24) Mu
̈
ller, H.; Bischoff, E.; Fiedler, V. B.; Weber, K.; Fugmann, B.;
Rosen, B. Ger. Offen. DE 4231289 A1, Mar 24, 1994.
The authors declare the following competing financial
interest(s): The authors submitted patent applications (PCT
Int. App. 2018, WO 2018110799 A1 20180621 and
KR2018068581 A 20180622) for this work.
ACKNOWLEDGMENTS
■
This work was supported by the National Research
Foundation of Korea Grants funded by the Korean Govern-
ment (Ministry of Science and ICT) (2017R1A2B2009393,
2018R1A4A1021703, and 2016R1A2A1A05005078).
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